The Study Of Freezing Inactivation Effect Of Profound Hypothermia On Osteosarcoma In Vitro

Objective: To study the histomorphologic change and death mechanism ofosteosarcoma cell after freezing inactivation of profound hypothermia onosteosarcoma in vitro and to probe the optimal way of the freezinginactivation effect.Methods: 1. group dividing: fresh specimens of osteosarcoma were dividedinto three groups:①group A (the control group).②group B(the cryotherapygroup with one freezing and thawing cycle).③group C (the cryotherapygroup with two freezing and thawing cycle). 2. Procedures: 4 specimens ofevery group B and C were collected and soaked into liquid nitrogen for5min,10min,15min,20min selectively and rewarming quickly. Group Cwere freezing and thawing twice. 3. Internal morphology and ultrasturcturechange of osteosarcoma cells after soaking of liquid nitrogen was observedby light and electron microscope and flow cytometry(FCM). Tumor celldeath and Apoptosis rate were calculated.Result: 1. specimens of group A(osteosarcoma cells) were observed by lightmicroscope and classified. It is suggested that some tumor cells with whole neclear become much more smaller than normal ones. 2. Necrosis rate was 42%～61% for group B and C, there was no statistical difference in group Band C. 3. Compared with group B, the tumor cells of group C weredamaged seriously. 4. Compared with group A, the apoptosis rate of groupB and C were significantly raising under examination with FCM. Apoptosisrate was 21%～48% for group B and C, there was no statistical differencein group B and C. Through observing the smaller cells, we come to find thetypical apoptosic phenomenon by transmission electron microscope.Conclusion: 1. Osteosarcoma cell deaths were included not onlydegeneration and necrosis but also apoptosis. Inreversible death was resultedfrom these two major inactivation effect. 2. Inactivation effect was reachedunder one single 5min freezing and thawing cycle. Physical and chemicalfactors were damaged worsely under twice freezing and thawing cycle, butthere were no significant raisings of apoptosis and necrosis rate comparedwith one sinle 5min cycle.